It was recently reported that 7 of the current top 8 selling drugs are proteins. However, it has also been noted that the cost of many protein-based drugs is extremely high, making their widespread use difficult to implement. To increase the utility of such molecules, it is essential that the cost of producing them be reduced. Protein conjugation is an essential feature of many protein-based drugs including antibody-drug conjugates and PEGylated proteins. Enzymatic methods offer tremendous potential to solve the problem of specificity and streamline the process of protein conjugation. The discovery that virtually any protein can be rendered a PFTase substrate by incorporation of a tetrapeptide recognition sequence coupled with the ability of the enzyme to tolerate a plethora of modifications within the isoprenoid structure has made PFTase an attractive choice for performing enzymatic protein labeling. In the previous funding period, we demonstrated that PFTase could be used to prepare site-selectively modified proteins, and employed this method to incorporate fluorescent labels and PEG groups into proteins for therapeutic applications that were evaluated in cell culture and animal models. A total of 21 papers and one patent from this work have been published with two more currently under review. In this next funding period, we hypothesize that PFTase-catalyzed enzymatic labeling will greatly facilitate the preparation of new ?biologics? for therapeutic applications by pursuing the following Aims: (1) Create mutant forms of PFTase that alter enzyme specificity and enlarge the size of the isoprenoid substrate that can be transferred. This will be pursued to generate a completely orthogonal form of PFTase that can transfer novel isoprenoids to specific proteins. Enlarging the isoprenoid binding site will allow efficient incorporation of larger moieties including whole polymer chains; (2) Prepare protein-polymer conjugates using polymer initiators installed on proteins via site-specific enzymatic labeling. Here, the standard approach of linking polymers to proteins will be reversed by incorporating site-selectively positioned initiators that can be used to grow polymeric chains of various types; (3) Use enzymatic protein labeling to prepare protein conjugates based on fibronectin scaffolds for imaging and therapeutic applications. Fibronectin (Fn) scaffolds that target EGF receptors on cancer cells will be modified with either DOTA ligands for PET imaging applications or drugs for therapeutic experiments in mouse xenograft models; (4) Employ triorthogonal PFTase substrates to create chemically self-assembled nanoring structures (CSANs) that incorporate protein toxins for therapeutic applications. Nanostructures functionalized with diphtheria toxin or perforin will be used to target CD3+ T-cells for the treatment of Type 1 Diabetes and other autoimmune disorders. Successful completion of these highly significant and innovative Aims could have a major impact in the field of protein conjugates and on their use in promoting human health.